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In this study, a titanium ion (Ti4+) functionalized polydopamine coated ferroferric oxide (Fe3O4@PDA@Ti4+) core-shell magnetic particle was prepared for the selective extraction of nucleotides. Firstly, different metal ions including Ti4+, Zr4+, Fe3+, Al3+, Cu2+, Zn2+, Ni2+ and Mg2+ were respectively immobilized onto Fe3O4@PDA particles and their extraction efficiency for five nucleotides [cytidine-5′-monophosphate (CMP), uridine-5′-monophosphate (UMP), guanosine-5′-monophosphate (GMP), thymidine-5′-monophosphate (TMP) and adenosine-5′-monophosphate (AMP)] were compared. Among these prepared materials, Fe3O4@PDA@Ti4+, which exhibited the highest extraction efficiency for nucleotides, was further characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy and energy dispersive X-ray spectroscopy. After being optimized of the extraction parameters including adsorbent amounts, extraction time, extraction temperature, type and concentration of the eluent, the prepared Fe3O4@PDA@Ti4+ magnetic particles were successfully applied for the selective extraction and determination of CMP, UMP, GMP, TMP and AMP in Cordyceps and Lentinus edodes. Good linearity (varying from 0.063 to 19.000 μg/mL, R2 > 0.999) and low limit of detection (LODs) (ranging between 0.0047 and 0.0141 μg/mL) for target analytes were achieved. These results demonstrated that the synthesized material in this study had potential for selective extraction of phosphorylated small molecular compounds in complicated matrix.

Background: As one of the bioactive components in Cordyceps sinensis (CS), proteins were rarely used as index components to study the correlation between the protein components and producing areas of natural CS. Objective: Protein components of 26 natural CS samples produced in Qinghai, Tibet, and Sichuan provinces were analyzed and compared to investigate the relationship among 26 different producing areas. Materials and Methods: Proteins from 26 different producing areas were extracted by Tris-HCl buffer with Triton X-100, and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Results: The SDS-PAGE results indicated that the number of protein bands and optical density curves of proteins in 26 CS samples was a bit different. However, the 2-DE results showed that the numbers and abundance of protein spots in protein profiles of 26 samples were obviously different and showed certain association with producing areas. Conclusions: Based on the expression values of matched protein spots, 26 batches of CS samples can be divided into two main categories (Tibet and Qinghai) by hierarchical cluster analysis. SUMMARY The number of protein bands and optical density curves of proteins in 26 Cordyceps sinensis samples were a bit different on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profilesNumbers and abundance of protein spots in protein profiles of 26 samples were obvious different on two-dimensional electrophoresis mapsTwenty-six different producing areas of natural Cordyceps sinensis samples were divided into two main categories (Tibet and Qinghai) by Hierarchical cluster analysis based on the values of matched protein spots. Abbreviations Used: SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2-DE: Two-dimensional electrophoresis, Cordyceps sinensis: CS, TCMs: Traditional Chinese medicines

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.

A magnetite@graphene oxide nanocomposite was first coated with polyethylenimine and then modified with phytic acid and titanium(IV) ions. The high loading with Ti(IV) and the good hydrophilicity of PEI and PA result in a material that can be applied to the efficient extraction of highly polar nucleobases, nucleosides and nucleotides. The physicochemical properties of the composite were investigated by scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, water contact angle measurements, thermogravimetric analysis, and vibrating sample magnetometry. A series of parameters that affect extraction and elution under the conditions of immobilized metal affinity chromatography (IMAC) and hydrophilic interaction liquid chromatography (HILIC) were examined. The analytes were eluted from the nanocomposites using 10 mM trisodium phosphate as the elution solution in the IMAC mode, and 50% methanol-water as elution solution in the HILIC mode. Figures of merit include (a) an intra-day precision of 0.1–1.0% in the IMAC mode; (b) an intra-day precision of 0.4%–0.8% in the HILIC mode; (c) detection limits between 1.8–2.8 ng mL−1 in the IMAC mode; and (d) detection limits of 4.0–10.5 ng mL−1 in the HILIC mode. The method was applied to the extraction of the nucleotides cytidine-5′-monophosphate (CMP), uridine-5′-monophosphate (UMP), guanosine-5′-monophosphate (GMP), and adenosine-5′-monophosphate (AMP), and the nucleobases and nucleosides hypoxanthine, adenosine, cytosine, inosine and cytidine from Cordyceps sinensis, Lentinus edodes and plasma samples.

An ion-pairing reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS) was developed for the determination of nucleotides, nucleosides and their transformation products in Cordyceps. Perfluorinated carboxylic acid, namely pentadecafluorooctanoic acid (PDFOA, 0.25mM), was used as volatile ion-paring agent and a reversed-phase column (Agilent ZORBAX SB-Aq column) was used for the separation of three nucleotides namely uridine-5'-monophosphate (UMP, 0.638-10.200microg/mL), adenosine-5'-monophosphate (AMP, 0.24-7.80microg/mL) and guanosine-5'-monophosphate (GMP, 0.42-13.50microg/mL), seven nucleosides including adenosine (0.55-8.85microg/mL), guanosine (0.42-6.75microg/mL), uridine (0.33-10.50micro/mL), inosine (0.21-6.60microg/mL), cytidine (0.48-15.30microg/mL), thymidine (0.20-6.30microg/mL) and cordycepin (0.09-1.50microg/mL), as well as six nucleobases, adenine (0.22-6.90microg/mL), guanine (0.26-4.20microg/mL), uracil (0.38-12.15microg/mL), hypoxanthine (0.13-4.20microg/mL), cytosine (0.39-12.45microg/mL) and thymine (0.26-8.25microg/mL) with 5-chlorocytosine arabinoside as the internal standard. The overall LODs and LOQs were between 0.01-0.16microg/mL and 0.04-0.41microg/mL for the 16 analytes, respectively. The contents of 16 investigated compounds in natural and cultured Cordyceps were also determined and compared after validation of the developed IP-RP-LC-MS method. The transformations of nucleotides and nucleosides in Cordyceps were evaluated based on the quantification of the investigated compounds in three extracts, including boiling water extraction (BWE), 24h ambient temperature water immersion (ATWE) and 56h ATWE extracts. Two transformation pathways including UMP-->uridine-->uracil and GMP-->guanosine-->guanine were proposed in both natural Cordyceps sinensis and cultured Cordyceps militaris. The pathway of AMP-->adenosine-->inosine-->hypoxanthine was proposed in natural C. sinensis, while AMP-->adenosine-->adenine in cultured C. militaris. However, the transformation of nucleotides and nucleosides was not found in commercial cultured C. sinensis.

Free and polymeric carbohydrates in Cordyceps, a valued edible mushroom and well-known traditional Chinese medicine, were determined using stepwise pressurized liquid extraction (PLE) extraction and GC-MS. Based on the optimized PLE conditions, acid hydrolysis and derivatization, ten monosaccharides, namely rhamnose, ribose, arabinose, xylose, mannose, glucose, galactose, mannitol, fructose and sorbose in 13 samples of natural and cultured Cordyceps were qualitatively and quantitatively analyzed and compared with myo-inositol hexaacetate as internal standard. The results showed that natural C. sinensis contained more than 7.99% free mannitol and a small amount of glucose, while its polysaccharides were usually composed of mannose, glucose and galactose with a molar ratio of 1.00:16.61~3.82:1.60~1.28. However, mannitol in cultured C. sinensis and cultured C. militaris were less than 5.83%, and free glucose was only detected in a few samples, while their polysaccharides were mainly composed of mannose, glucose and galactose with molar ratios of 1.00:3.01~1.09:3.30~1.05 and 1.00:2.86~1.28:1.07~0.78, respectively. Natural and cultured Cordyceps could be discriminated by hierarchical clustering analysis based on its free carbohydrate contents.

A HPLC coupled with diode array detection (DAD) and evaporative light scattering detection (ELSD) method for qualitative and quantitative analysis of eight nucleosides and nucleobases, three carbohydrates and myriocin in Cordyceps was developed. A Prevail Carbohydrate ES column was employed for the separation within 50 min. Nucleosides and their bases were tested at UV 254 nm. ELSD was connected with DAD to determine myriocin and carbohydrates. The optimum drift tube temperature of ELSD was at 94°C with the nitrogen flow rate of 2.0 L/min. All calibration curves showed good linearity (R2>0.9933) during the test ranges. The precision, repeatability, accuracy, LOD and LOQ were also fully investigated. This developed method was successfully applied to quantify 12 components, eight nucleosides and nucleobases, three carbohydrates and myriocin, in natural and cultured Cordyceps, which provides another view for quality control of Cordyceps sinensis.

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.

Sample preparation is the first and very important step, which can greatly influence the repeatability and accuracy of the analysis. To date, several sample preparation methods with different solvents have been used for quantitative determination of nucleosides in Cordyceps, but their data are greatly various. In this study, five nucleosides, including adenosine, guanosine, inosine, uridine and cordycepin, in Cordyceps were determined using three extraction methods i.e. organic solvent pressurized liquid extraction, boiling water extraction and ambient temperature water extraction and high performance liquid chromatography (HPLC)-diode array detection (DAD). The similar results were obtained when organic solvent pressurized liquid extraction and boiling water extraction were applied. However, the amounts of nucleosides in natural C. sinensis and cultured C. militaris extracted with ambient temperature water were greatly increased except those of adenosine in natural C. sinensis and cordycepin in cultured C. militaris. In addition, the amount of investigated nucleosides in cultured C. sinensis had no obvious variation among the three extraction methods. The results suggest that sample preparation has significant effect on the quantification of nucleosides in Cordyceps.

A method based on optimum acid hydrolysis followed by high-performance liquid chromatography (HPLC) with diode array detection was developed for quantitative determination of bio-available nucleosides, present as purine and pyrimidine bases including adenine, cytosine, guanine, hypoxanthine, thymine and uracil, in natural and cultured Cordyceps . It was found that the optimum conditions was hydrolyzing Cordyceps sample in eight folds of pure commercial perchloric acid for 1 h at 95–100 °C. The determination was achieved by using a Zorbax SB-AQ analytical column (250 mm × 4.6 mm i.d., 5 μm) at gradient elution with diode-array detection. All calibration curves showed good linearity ( r 2 > 0.999) within test ranges. The developed method showed good repeatability for the quantification of six investigated nucleobases in Cordyceps with intra- and inter-day variations of less than 9.0 and 9.1%, respectively. The validated method was successfully applied to quantify bio-availbale nucleosides in natural and cultured Cordyceps , which is helpful to control their quality.

Cordyceps sinensis, a well-known and valued traditional Chinese medicine, is also called DongChongXiaCao (winter worm summer grass) in Chinese. It is commonly used to replenish the kidney and soothe the lung for the treatment of fatigue, night sweating, hyposexualities, hyperglycemia, hyperlipidemia, asthemia after severe illness, respiratory disease, renal dysfunction and renal failure, arrhythmias and other heart disease, and liver disease. As the rarity and upstanding curative effects of natural Cordyceps, several mycelial strains have been isolated from natural Cordyceps and manufactured in large quantities by fermentation technology, and they are commonly sold as health food products in Asia. In addition, some substitutes such as Cordyceps militaris also have been used and adulterants also confused the market. Therefore, quality control of C. sinensis and its products is very important to ensure their safety and efficacy. Herein, markers and analytical methods for quality control of Cordyceps were reviewed and discussed.

A simple and rapid pressurized liquid extraction (PLE) and high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) method was developed for qualitative and quantitative determination of nucleosides, bases and their analogues in natural and cultured Cordyceps. The samples were extracted using PLE. The separation was achieved on a ZORBAX Eclipse XDB-C18 column with gradient elution of methanol and 5 mM aqueous ammonium acetate as mobile phase. Target compounds were identified by characterizing their product ions, precursor ions and retention times. Quantitative analysis of investigated compounds were performed using time programmed selective ion monitoring (SIM) or selective reaction monitoring (SRM) with 10 segments in positive (negative for uridine) ion mode. The results showed that 43 bases, nucleosides and their analogues were detected in Cordyceps, of these 16 compounds were identified. The simultaneous determination of seven nucleosides and six bases in Cordyceps was achieved using PLE and HPLC–ESI–MS/MS method described above, which afforded good linearity, selectivity, precision, recovery, short analysis time as well as LOD and LOQ in the ng/ml range.

A HPLC coupled with diode array detection (DAD) and evaporative light scattering detection (ELSD) method for qualitative and quantitative analysis of eight nucleosides and nucleobases, three carbohydrates and myriocin in Cordyceps was developed. A Prevail Carbohydrate ES column was employed for the separation within 50 min. Nucleosides and their bases were tested at UV 254 nm. ELSD was connected with DAD to determine myriocin and carbohydrates. The optimum drift tube temperature of ELSD was at 94 degrees C with the nitrogen flow rate of 2.0 L/min. All calibration curves showed good linearity (R(2)0.9933) during the test ranges. The precision, repeatability, accuracy, LOD and LOQ were also fully investigated. This developed method was successfully applied to quantify 12 components, eight nucleosides and nucleobases, three carbohydrates and myriocin, in natural and cultured Cordyceps, which provides another view for quality control of Cordyceps sinensis.

In present study, a capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine, guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2'-deoxyuridine, uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside as an internal standard (IS). The CE separation conditions and MS parameters were optimized systematically for achieving good CE resolution and MS response of the investigated compounds. The optimum CE electrolyte was 100 mM formic acid containing 10% (v/v) methanol. The optimum MS parameters were as follows: 75% (v/v) methanol containing 0.3% formic acid with a flow rate of 3 microL/min was selected as the sheath liquid; the flow rate and temperature of drying gas were 6 L/min and 350 degrees C, respectively. The optimized CE-MS method was successfully applied for the simultaneous determination of 12 nucleosides and nucleobases in natural and cultured Cordyceps. On the basis of quantitative results, the total content of nucleosides is much higher in cultured Cordyceps (9138+/-4823 microg/g for cultured C. sinensis; 3722+/-1446 microg/g for C. militaris) than in natural ones (2167+/-412 microg/g). However, the hypoxanthine (131+/-47 microg/g) and inosine (335+/-90 microg/g) are much higher in natural C. sinensis. Cordycepin, which is abundant in cultured C. militaris (2276.5+/-842.6 microg/g), is only found in natural C. sinensis with very low content and cannot be detected in the cultured ones.

Ten free fatty acids namely lauric acid, myristic acid, pentadecanoic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, docosanoic acid and lignoceric acid and four free sterols including ergosterol, cholesterol, campesterol and beta-sitosterol in natural (wild) Cordyceps sinensis, Cordyceps liangshanensis and Cordyceps gunnii, as well as cultured C. sinensis and Cordyceps militaris were first determined using pressurized liquid extraction (PLE), trimethylsilyl (TMS) derivatization and GC-MS analysis. The conditions such as the amount of reagent, temperature and time for TMS derivatization of analytes were optimized. Under the optimum conditions, all calibration curves showed good linearity within the tested ranges. The intra- and inter-day variations for 14 investigated compounds were less than 3.4% and 5.2%, respectively. The results showed that palmitic acid, linoleic acid, oleic acid, stearic acid and ergosterol are main components in natural and cultured Cordyceps which could be discriminated by hierarchical clustering analysis based on the contents of 14 investigated compounds or the 4 fatty acids, where the contents of palmitic acid and oleic acid in natural Cordyceps are significantly higher than those in the cultured ones.

Safety and efficacy are two key important aspects for the quality assessment of health-food products. Cordyceps sinensis, which has multiple pharmacological activities, is one of the most expensive traditional Chinese medicines (TCMs) and is used frequently in health-food products in China. Usually, nucleosides and their related compounds have been used as markers for evaluation of the quality of natural and/or cultured Cordyceps. However, heavy metals should also be considered as they may affect the safety of clinical use. In the present study, the contents of six nucleosides (adenosine, cytidine, guanosine, uridine, inosine and thymidine), six nucleobases (adenine, cytosine, guanine, uracil, hypoxanthine and thymine) and five nucleotides (adenosine-5′-monophosphate (AMP), cytidine-5′-monophosphate (CMP), guanosine-5′-monophosphate (GMP), uridine-5′-monophosphate (UMP), and thymidine-5′-monophosphate (TMP)) in natural C. sinensis samples from different collecting places were simultaneously determined by a validated HPLC-DAD method. At the same time, six heavy metals including Hg, As, Cr, Cd, Cu and Pb in natural C. sinensis and their related soil samples were determined by atomic absorption spectrometry (AAS). The results showed that the total content of nucleosides and their related compounds, as well as the individual levels of nucleotides, are much higher in fresh samples (collected in 2012) as compared to stored samples (collected in 2011). Therefore, the storage conditions may affect on the quality of natural C. sinensis. On the other hand, the total and individual contents of heavy metals vary in different Cordyceps and soil samples, but the levels of As and Cu in Cordyceps are correlated well with the soil samples from their collection locations. Therefore, the high levels of heavy metals, especially As and Cu, in natural Cordyceps originate from the soil of the producing areas.

Determination of myriocin-like long-chain bases (MLBs) is important for physiological and pharmacological studies of Cordyceps. Herein, a simple and sensitive reversed-phase liquid chromatographic method, based on the pre-column derivatization with O-phthalaldehyde, was developed for the simultaneous determination of four MLBs, namely myriocin, phytosphingosine, sphingosine and sphinganine in Cordyceps. The chromatographic and derivatization conditions were intensively optimized, and the structures of the derivatives were confirmed by mass spectra. The developed method was applied for the analysis of four MLBs in C. sinensis, C. militaris, C. cicadae and C. gunnii and cultured C. cicadae, which also could be used for the fast determination of the MLBs in pharmaceutical products and biological fluids.